HK1072948B - Ifnar2 mutants, their production and use - Google Patents

Description

IFNAR2 mutant, its manufacture and use

Technical Field

The present invention relates to mutant polypeptides of the beta chain of the type I interferon receptor (MIFNAR2) having enhanced affinity for interferon-beta, which prolong the in vivo effects of IFN-beta as compared to the wild-type protein.

Background

The types of interferons are classified as type I interferons produced by leukocytes and fibroblasts, and type II interferons induced by mitogens or "immune" (Pestka et al, 1987). Type I interferons include interferon alpha (IFN- α), interferon beta (IFN- β), and interferon omega (IFN- ω), as analyzed for sequence identity and common biological activity; while type II interferons include interferon gamma (IFN- γ).

IFN-alpha, IFN-beta and IFN-omega genes are clustered on the short arm 25 of chromosome 9 (lencyl, 1982). There are at least 25 non-allelic IFN-alpha genes, 6 non-allelic IFN-omega genes and 1 IFN-beta gene. Are believed to have evolved from a common ancestral gene. Within these classes, the IFN- α genes have at least 80% sequence identity to each other; IFN- β genes have about 50% sequence identity to IFN- α; the IFN-omega gene is 70% homologous to IFN-alpha (Weissman et al, 1986; Dron et al, 1992) with an IFN-alpha molecular weight range of 17-23kd (165-166 amino acids); IFN- β is about 23kd (166 amino acids) and IFN- ω is about 24kd (172 amino acids).

Type I interferons are pleiotropic cytokines that have host antiviral and parasite defensive activity, anti-cancer properties and are useful as immunomodulators (Baron et al, 1994; Baron et al, 1991). The physiological responses of type I interferons include: anti-proliferative activity of normal and transformed cells, stimulating cytotoxic activity of lymphocytes, natural killer cells and phagocytes, modulating cell differentiation, stimulating MHC class I antigen expression, inhibiting MHC class II and modulating various cell surface receptors. Under normal physiological conditions, most human cells constitutively secrete IFN- α and IFN- β (IFN α/β) at low levels; the expression of infectious agents (viruses, bacteria, mycoplasma and protists), dsRNA, and cytokines (M-CSF, IL-1. alpha., IL-2, TNF. alpha.) can be upregulated by the addition of various inducers. The in vivo effects of type I interferons can be monitored using the surrogate markers neopterin, 2 ', 5' oligoadenylate synthetase and beta 2 microglobulin (Alam et al, 1997; Fierlbeck et al, 1996; Salmonon et al, 1996).

Type I interferenceThe hormone (IFN α/β/ω) acts through cell surface receptor complexes, inducing specific biological effects such as antiviral, antitumor and immunomodulatory effects. Type I IFN receptors (IFNARs) are hybrid multimeric receptor complexes composed of at least two distinct polypeptide chains (Colamonici et al, 1992; Colamonici et al, 1993; Platanias et al, 1993). The genes encoding these chains are found on chromosome 21, and their proteins are expressed on the surface of most cells (Tan et al, 1973). The two receptor chains were initially designated α and β, followed by a renaming of the α subunit as IFNAR1 and the β subunit as IFNAR 2. In most cells, IFNAR1 (alpha subunit, Uze subunit) (Uze et al, 1990) has a molecular weight of 100-130 kd; and IFNAR2 (beta chain, beta)_LIFN alpha/beta R) has a molecular weight of 100 kd. In certain cell types (monocyte cell lines and normal bone marrow cells), another receptor complex has been identified in which IFNAR2 subunit (β)_S) Expressed as a truncated receptor with a molecular weight of 51 kd. IFNAR1 and IFNAR2 beta have been cloned_SAnd beta_LSubunits (Novick et al, 1994; Domanski et al, 1995). IFNAR2 beta_SAnd beta_LSubunits have identical extracellular and transmembrane domains, but their cytoplasmic domains are identical for only the first 15 amino acids. IFNAR2 subunit alone can bind IFN alpha/beta, and IFNAR1 subunit can not bind IFN alpha/beta. When the human IFNAR1 receptor subunit alone was transfected into mouse L-929 fibroblasts, no human IFN α other than IFN α 8/IFN α B could bind to the cells (Uze et al, 1990). In the absence of human IFNAR1 subunit, L cells transfected with human IFNAR2 subunit can bind human IFN alpha, binding force of about 0.45nM Kd. Human IFNAR2 subunit was transfected in the presence of human IFNAR1 subunit and was shown to bind with high affinity with a Kd of 0.026-0.114nM (Novick et al, 1994; Domanski et al, 1995). It is estimated that most cells have 500-20,000 high affinity and 2,000-100,000 low affinity IFN binding sites. Although IFNAR1/2 complex (alpha/beta)_ＳOr alpha/beta_L) The subunit binds IFN alpha with high affinity, but only alpha/beta_LThis pair appears to be functional signaling receptors.

IFNAR1 and IFNAR2 beta_LSubunit transfection into mouse L-929 cells, and then with IFN alpha 2 incubation, can induce antiviral state, startIntracellular proteins are phosphorylated, leading to activation of intracellular kinases (Jak1 and Tyk2) and transcription factors (STAT1, 2 and 3) (Novick et al, 1994; Domanski et al, 1995). In a corresponding experiment, IFNAR2 beta was transfected_LSubunits are unable to initiate similar reactions. Therefore the production of functional activity (antiviral response) maximum induction needs IFNAR2 beta_LThe subunit is associated with IFNAR1 subunit.

In addition to the membrane-bound cell surface IFNAR forms, soluble IFNAR has been identified in human urine and serum (Novick et al, 1994; Novick et al, 1995; Novick et al, 1992; Lutfalla et al, 1995). On SDS-PAGE, the apparent molecular weight of soluble IFNAR isolated from serum was 55kd, whereas the apparent molecular weight of urine-soluble IFNAR was 40-45kd (p 40). Soluble p40IFNAR2 exists as an mRNA transcript containing almost the entire extracellular domain of IFNAR2 subunit with two additional amino acids at the carboxy terminus. The soluble IFNAR2 receptor has 5 potential glycosylation sites. Soluble p40IFNAR2 has been shown to bind IFN alpha 2 and IFN beta, and inhibit IFN alpha class mixture ("leukocyte IFN") and various type I IFN in vitro antiviral activity (Novick et al, 1995). Recombinant IFNAR2 subunit Ig fusion proteins have been shown to inhibit various type I IFNs (IFN. alpha.A, IFN. alpha.B, IFN. alpha.D, IFN. beta., IFN. alpha. Con1 and IFN-. omega.) and Daudi cells and alpha/beta_sSubunit binding of double transfected COS cells.

Type I IFN signaling pathways have been identified (Platanias et al, 1996; Yan et al, 1996; Qureshi et al, 1996; Duncan et al, 1996; Sharf et al, 1995; Yan et al, 1996). It is thought that the initiation process leading to signaling occurs when IFN α/β/ω binds to IFNAR2 subunit, whereupon IFNAR1 subunit reassociates to form the IFNAR1/2 complex (Platanias et al, 1994). IFN α/β/ω binds to IFNAR1/2 complex, resulting in the activation of two Janus kinases (Jak1 and Tyk2) that are believed to phosphorylate specific tyrosines on IFNAR1 and IFNAR2 subunits. Once these subunits are phosphorylated, STAT (STAT1, 2, and 3) molecules are phosphorylated, resulting in dimerization of the STAT transcription complex, which then translocates to the nucleus, activating specific IFN inducible genes.

A randomized double-blind placebo-controlled two-year multicenter study demonstrated that Intrathecal (IT) injection of natural human fibroblast interferon (IFN β) was effective in reducing exacerbations of exacerbation-remitting Multiple Sclerosis (MS). In this study, the mean decrease in the rate of exacerbation was significantly higher in 34 MS patients receiving intrathecal IFN β treatment than in 35 placebo control patients (Jacobs et al, 1987).

The pharmacokinetics and pharmacodynamics of type I IFN in humans were evaluated (Alan et al, 1997; Fierlbeck et al, 1996; Salmonon et al, 1996). IFN beta clearance is relatively fast, so its bioavailability is lower than most cytokines expected. Although the pharmacodynamics of IFN β in humans has been evaluated, no clear correlation between IFN β bioavailability and clinical efficacy has been determined. In normal healthy volunteers, one intravenous injection (iv) of a bolus of recombinant CHO-produced IFN β (6MIU) resulted in a rapid phase distribution of 5 minutes with a terminal half-life of about 5 hours (Alam et al, 1997). After subcutaneous (sc) or intramuscular (im) injection of IFN β, serum levels were only maintained at about 15% of those for systemic administration. After Iv, im or sc injection, the potency of IFN β (measured AS a change in 2 ' 5/-oligoadenylate synthetase (2 ', 5 ' -AS) in PBMC) increased in the first 24 hours and slowly decreased to baseline levels in the following 4 days. Regardless of the route of administration, the extent and duration of the biological effect is the same.

Multiple dose pharmacodynamic studies of IFN β were performed in human melanoma patients (Fierlbeck et al, 1996), with IFN β administered by the sc route, 3MIU doses, 3 times per week for 6 months. After the second injection (4 days), pharmacodynamic markers: 2 ', 5' -AS synthetase, beta₂Activation of microglobulin, neopterin and NK cells peaked, declined after 28 days, and remained only mildly elevated up to 6 months.

The extracellular portion of human IFNAR2 (IFNAR2-EC) was expressed in E.coli, purified and refolded, and its characteristic of interacting with interferon alpha (IFN. alpha.2) was reported (Piehler and Schreiber 1999A). The 25kd of non-glycosylated IFNAR2-EC was shown to be a inhibition of IFN alpha 2 antiviral activity with full activity of stable protein. Through gel filtration, chemical cross-linking and solid phase detection determination, combined IFN alpha 2 stoichiometry is 1: 1. The affinity of this interaction was about 3nM (Piehler and Schreiber 2001). The rate of complex formation is higher than other cytokine-receptor interactions. The salt dependence of the binding kinetics suggests that electrostatic forces have a limited but significant effect on the complex formation. As the protonation pH of the base decreases, the dissociation constant increases to pKa 6.7. IFN beta IFNAR2 affinity than IFN alpha 2 IFNAR2 affinity is about 2 times higher (Piehler and Schreiber, 1999B).

A single mutation in the IFNAR2 binding site was analyzed to map the binding differential between IFN α 2 and IFN β (Piehler and Schreiber, 1999B). For example, the mutation H78A was found to stabilize the complex with IFN beta about 2-fold, and IFN alpha 2 complex is unstable more than 2-fold. It was found that the mutation N100A hardly influences the rate of IFN alpha 2 binding, but reduces the IFN beta dissociation rate constant about 4 times.

European patent EP1037658 describes that administration of interferon in the form of an IFN-binding chain complex containing the human IFN alpha/beta receptor (IFNAR) which acts as a carrier protein for IFN prolongs the in vivo effects of type I Interferon (IFN). The complex also improves the stability of the IFN and increases the activity of the IFN. Such complexes can be non-covalent complexes, or complexes in which IFN and IFNAR are covalently or peptide-linked. EP1037658 also states that storing IFN in such a complex increases the shelf life of the IFN and allows storage under warmer conditions than was previously not possible.

There is therefore a need for IFN beta instead of IFN alpha 2 with improved affinity IFNAR2, and preparation of IFN beta better and specific carrier IFNAR2 needs.

Summary of The Invention

The present invention provides an IFNAR2 mutant polypeptide (MIFNAR2), or homologue, functional derivative, fusion protein or salt thereof, having a higher affinity for interferon beta (IFN β) than its wild type, mutated at amino acid residue 78, histidine and asparagine at position 100. These mutations are amino acid substitutions, preferably with conservative amino acids, more preferably alanine, aspartic acid and histidine. The IFNAR2 mutant has about 25 times, preferably 50 times, more preferably 100 times higher affinity than wild type, preferably about 30 nM. More specifically, the invention provides IFNAR2 mutant polypeptide fragments comprising an extracellular domain. In addition, the present invention provides a DNA encoding the IFNAR2 mutant polypeptide, a vector containing the DNA, a host cell containing the vector, and a method for producing the mutant polypeptide of the present invention by culturing the host cell and isolating the produced mutant polypeptide.

In another aspect, the invention provides the use of an IFNAR2 mutant polypeptide in the manufacture of a medicament for modulating the effects of IFN in vivo, preferably the effects of IFN β.

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an IFNAR2 mutant or extracellular domain fragment thereof, administered alone or in combination with an IFN, more preferably IFN β, either separately or covalently. More specifically, the pharmaceutical compositions provided by the present invention promote the antiviral, anticancer and immunomodulatory properties of IFN β and treat autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, ulcerative colitis and lupus.

In addition, the present invention provides methods of treating autoimmune diseases, viral diseases, and cancer comprising administering the IFNAR2 mutant polypeptides of the invention.

In addition, the present invention provides the use of IFNAR2 mutant polypeptides, preferably administered with an IFN antagonist, to inhibit IFN activity in diseases caused or exacerbated by IFN.

The invention also provides the use of the IFNAR2 mutant polypeptides of the invention in a preparation to prevent oligomerization of IFNs.

Brief description of the drawings

FIG. 1 (left) the stimulated binding and free IFN β, calculated according to the law of mass action, with a constant concentration of IFN (50pM) and increasing concentrations of IFNAR2-EC (left) and mutant IFNAR2-EC (right) kd 3nM and 50pM, respectively.

Figure 2 shows the amino acid sequence of the extracellular domain of IFNAR2 protein (excluding the leader sequence), and modified amino acid residues (marked with asterisks).

Figure 3 shows IFN beta and IFN alpha 2 and IFNAR2-EC H78A/N100A mutant binding. The reflection interference spectrophotometry (rifS) was used to determine the IFN beta and IFN alpha 2 and wild type IFNAR2-EC (upper panel), IFNAR2-ECH78A/N100A mutant (middle panel) binding and dissociation; wild type and mutant IFNAR2-EC H78A/N100A with IFN beta binding (lower panel), IFNAR2 fixed to the surface (see Piehler and Schreiber, 2001). The Y-axis is the signal (nm) and the X-axis is the time (sec).

Figure 4 shows that IFN beta by IFNAR2 wild type and mutant binding. A constant amount of IFN β (10pM) was mixed with different concentrations of IFNAR2(R2) wild-type and mutant (single mutant R2N100A and R2H 78A; double mutant R2H78A/N100A, R2H78A/N100H and R2H78A/N100D) and the residual antiviral activity in WISH cells at equilibrium was determined. In the upper box, showing in the absence of IFNAR2 IFN beta antiviral map for its concentration content (Y ═ survival index). This figure can be used as a standard to determine how much IFN beta is free (active) in this antiviral assay.

Detailed Description

The present invention relates to a mutant of the beta chain of the type I interferon receptor (MIFNAR2) (MIFNAR2) with enhanced affinity for interferon beta rather than IFN alpha 2, which is mutated at amino acid residues H78 and N100 (see fig. 2 DNA sequence of wild-type IFNAR 2). The invention also relates to a drug carrier system containing the extracellular domain (EC) of MIFNAR2, which enhances the IFN activity. The present invention relates to mifmar 2, or an analogue, functional derivative, fusion protein, fragment or salt thereof.

The carrier is typically administered to prolong the retention time of proteins with molecular weights below 50,000D (e.g., interferon) in the vesicles. A particular advantage is that non-covalent association with the carrier results in constant release of the drug. It is desirable to use such carriers to utilize a fraction of the free drug's therapeutic activity at any time (about 20%) and to have an amount of drug bound to the carrier that is preserved (about 80%).

FIG. 1 (left panel) illustrates the simulated binding and free IFN β concentration (measured by reflectance interference spectrophotometry [ rifS ]) in the presence of different concentrations of IFNAR2 based on the law of mass action and 3nM kd. This excitation shows that in order to achieve 20% free IFN beta (10pM, equal to about 100 units) and 80% binding, for example 12.5nM of IFNAR2 protein (equal to 300. mu.g/kg of non-glycosylated IFNAR2) is required at very high concentration. Therefore, the use of IFN beta affinity 50 times higher IFNAR2 mutants as a carrier (see figure 1 simulation, right panel) has advantages, because such mutants theoretically only need about 0.24nM to reach 20% of the free IFN beta (equal to 6 u g/kg).

An IFNAR2 mutant with enhanced affinity for IFN was generated (MIFNAR 2). To obtain mifpar 2-EC, two amino acid residues of wild-type IFNAR2-EC (fig. 1, seq id no1) were modified: histidine at residue 78, and asparagine at residue 100 (see FIGS. 2, 3 and 4, SEQ ID NOS: 2, 3 and 4). This mutant IFNAR2-EC protein proved to be a better specific carrier of IFN beta, i.e. the affinity for IFN beta improved and IFN alpha 2 affinity remains unchanged. These mutants were found to have 26, 40 and about 50 times higher affinities for IFN β than the wild type (table 4). The results obtained show that, despite the increased affinity of the mutant soluble receptor (kd of H78A/N100A IFNAR2 mutant is about 30pM, whereas kd of the wild-type protein is 3nM), sufficient IFN β remains unbound to exert therapeutic activity, as demonstrated by the antiviral protection of VSV against WISH cells (fig. 4). The results also show, with low concentrations of IFNAR mutant EC, can achieve and wild type IFNAR2EC combined with IFN levels (equilibrium state combined IFN). The best results were obtained with mutants modified with both residues, in particular with both amino acids mutated to alanine, H78A/N100A IFNAR2, for example, only 30 times less H78A/N100A IFNAR mutant than the wild-type IFNAR2 protein is required to achieve 80% bound IFN β (8pM bound, 2pM free IFN β).

This result shows, double mutation of IFNAR2 can be more effective in binding IFN beta, and only need a lower amount to complete its carrier IFN beta function.

The advantages of using MIFNAR2-EC are: (I) lower amounts (technically feasible) of this receptor can be administered as a vehicle, (II) some of the adverse side effects of interferon therapy can be reduced because the mutant activity is stable and the amount of IFN β administered can be reduced, (III) the mutant activity is specific for IFN β, and (IV) in certain inflammatory diseases, it may be desirable to reduce the concentration of IFN in certain cases where the mutant is employed as a specific and effective antagonist against IFN β rather than IFN α 2.

MIFNAR2-EC may be administered alone to stabilize and enhance the activity of endogenous IFN β, which is particularly useful in treating patients with diseases caused by elevated levels of natural IFN such that IFN already circulating in the body exerts its natural effect in combating such diseases. MIFNAR2-EC will have a specific effect on endogenous IFN β and a slight effect on IFN α 2. Alternatively, MIFNAR2-EC may be co-administered with IFN, preferably with IFN- β, or may be administered covalently in conjunction with IFN- β to modulate the activity of IFN- β. The mifmar 2 and IFN β used to produce the complex are preferably recombinant molecules.

The technology required to produce mutant EC IFNAR2 and IFN fusion proteins is similar to the technology detailed in WO9932141 for producing wild-type IFNAR/IFN complexes, in which the wild-type version is replaced by mutant IFNAR2(MIFNAR2) at positions H78 and N100.

The use of the MIFNAR2/IFN beta non-covalent binding complexes of the invention suggests that the required low concentrations of IFNAR2-EC may be used in the treatment of a variety of conditions in which IFN itself has therapeutic effect.

These diseases include diseases in which free IFN has been shown to have certain therapeutic effects, such as antiviral, anticancer and immunomodulatory effects. This mutant IFNAR2/IFN complex is expected to be more effective in treating viral, oncogenic, and autoimmune diseases by virtue of its greater potency, enhanced activity, and/or improved pharmacokinetics (i.e., half-life).

When administered in vivo, the interferon receptor complex enhances the bioavailability, pharmacokinetics, and/or pharmacodynamics of the IFN, thereby enhancing the antiviral, anticancer, and immunomodulatory properties of the IFN.

Preferred molecules for use in the complexes of the invention comprise the amino acid sequences of native IFN β and MIFNAR2 (SEQ ID NOs: 2, 3 and 4). The natural sequence is a naturally occurring human IFN beta. These sequences are known and can easily be found in the literature. Naturally occurring allelic variations are also considered to be native sequences.

The invention also relates to congeners of the MIFNAR 2-EC. Such homologues may be homologues of up to 30, preferably up to 20, most preferably 10 amino acid residues of the protein deleted, added or substituted by other residues, except for mutations at residues 78 and 100 which may result in a reduction in the affinity of mifmar 2 for IFN β and wild type IFNAR2 for IFN β. These analogs can be prepared using known synthetic and/or site-directed mutagenesis techniques, or any other known suitable technique.

The congener preferably has a substantially repeated amino acid sequence of mifpar 2 and thus has similar activity. Thus, it can be determined whether any given analog has substantially similar activity and/or stability to the proteins and complexes of the invention by routine experimentation, including binding of each analog and determination of biological activity. MIFNAR2-EC congeners bind IFN beta with at least 15-fold, about 50-100-fold greater affinity than wild-type protein, without significant change in the affinity for IFN alpha 2. MIFNAR2-EC congeners can exhibit a kd of about 30pM or less for IFN β. Binding assays for the interaction of MIFNAR2 and IFN may include analytical gel filtration, heterogeneous light detection (such as surface plasmon resonance [ SPR ], or reflectance interference spectrophotometry [ rifS ], which is similar to the widely used BIACORE technique), and fluorescence spectrophotometry (Piehler and Schreiber, 1999A; Piehler and Schreiber, 2001).

Analogs of such complexes, or encoding nucleic acid sequences thereof, useful in the present invention comprise a limited set of corresponding sequences, such as alternative peptides or polynucleotides, which are routinely obtained by one of ordinary skill in the art without undue experimentation in light of the description and guidance provided herein. For a detailed description of the protein chemistry and structure, see Schulz et al, the principles of protein structure, Springer Verlag, New York (1978); and Creighton, t.e., protein: structural and molecular properties, w.h. freeman & Co, San Francisco (1983), incorporated herein by reference.

For a description of nucleotide sequence substitutions, e.g., codon identity, see Ausubel et al (1987, 1992), A.1.I-A.1.24 and Sambrook et al (1987, 1992), 6.3 and 6.4, see appendix C and D.

Preferred changes in the congeners of the invention are so-called "conservative" substitutions. Those conservative amino acid substitutions in the protein sequence of the invention include synonymous amino acids with similar physicochemical properties in a group, and substitutions between members of the group will preserve the biological function of the molecule (Grantham, 1974). It is known that insertions or deletions can be made in the above-mentioned sequences without altering their function, in particular if such insertions or deletions involve only a few amino acids, for example less than 30, preferably less than 10, without removing or replacing amino acids which are critical for the functional conformation, for example cysteine residues (Anfinsen, 1973). Congeners produced by such deletions or insertions are within the invention. Preferred groups of synonymous amino acids are those defined in Table II, and most preferred groups of synonymous amino acids are those defined in Table III.

TABLE 1 grouping of synonymous amino acids

Synonymous group of amino acids

Ser Ser，Thr，Gly，Asn

Arg Arg，Gln，Lys，Glu，His

Leu Ile，Phe，Tyr，Met，Val，Leu

Pro Gly，Ala，Thr，Pro

Thr Pro，Ser，Ala，Gly，His，Gln，Thr

Ala Gly，Thr，Pro，Ala

Val Met，Tyr，Phe，Ile，Leu，Val

Gly Ala，Thr，Pro，Ser，Gly

Ile Met，Tyr，Phe，Val，Leu，Ile

Phe Trp，Met，Tyr，Ile，Val，Leu，Phe

Tyr Trp，Met，Phe，Ile，Val，Leu，Tyr

Cys Ser，Thr，Cys

His Glu，Lys，Gln，Thr，Arg，His

Gln Glu，Lys，Asn，His，Thr，Arg，Gln

Asn Gln，Asp，Ser，Asn

Lys Glu，Gln，His，Arg，Lys

Asp Glu，Asn，Asp

Glu Asp，Lys，Asn，Gln，His，Arg，Glu

Met Phe，Ile，Val，Leu，Met

Trp Trp

TABLE 2

More preferred grouping of synonymous amino acids

Synonymous group of amino acids

Ser Ser

Arg His，Lys，Arg

Leu Leu，Ile，Phe，Met

Pro Ala，Pro

Thr Thr

Ala Pro，Ala

Val Val，Met，Ile

Gly Gly

Ile Ile，Met，Phe，Val，Leu

Phe Met，Tyr，Ile，Leu，Phe

Tyr Phe，Tyr

Cys Cys，Ser

His His，Gln，Arg

Gln Glu，Gln，His

Asn Asp，Asn

Lys Lys，Arg

Asp Asp，Asn

Glu Glu，Gln

Met Met，Phe，Ile，Val，Leu

Trp Trp

TABLE 3

Most preferred grouping of synonymous amino acids

Synonymous group of amino acids

Ser Ser

Arg Arg

Leu Leu，Ile，Met

Pro Pro

Thr Thr

Ala Ala

Val Val

Gly Gly

Ile Ile，Met，Leu

Phe Phe

Tyr Tyr

Cys Cys，Ser

His His

Gln Gln

Asn Asn

Lys Lys

Asp Asp

Glu Glu

Met Met，Ile，Leu

Trp Met

Examples of generating amino acid substitutions that may be used to obtain the MIFNAR2 or MIFNAR2-EC congener proteins used in the present invention include U.S. Pat. No. RE 33,653 to Mark et al; 4,959,314; 4,588,585 and 4,737,462; 5, 116,943 to Koths et al; 4,965,195 granted to Namen et al; known method steps described in U.S. Pat. No. 5,017,691 to Lee et al, and lysine-substituted proteins described in U.S. Pat. No. 4,904,584 to Shaw et al.

The term "substantially corresponds to" refers to the same class comprising sequences with only minor changes compared to the sequence of the basic mifpar 2 or mifpar 2-EC, without affecting its basic characteristics, such as its specifically enhanced binding affinity to IFN β. The types of changes that are generally considered to be "substantially corresponding" to this term are those that result in several minor modifications of the DNA encoding the complex of the invention using conventional mutagenesis techniques and are screened for changes in the desired activity in the following manner.

The mifmar 2 portion of the complex preferably has a core sequence identical to the native sequence or an active fragment thereof, or a variant having an amino acid sequence which is at least 70% identical to the native amino acid sequence and retains its biological activity. More preferably, the sequence has at least 85% identity, at least 90% identity, or most preferably at least 95% identity to the native sequence.

With respect to the IFN portion of the complex, the core sequence that can be employed is a native sequence, or a biologically active fragment thereof, or a variant having a sequence at least 70% identical, more preferably at least 85% identical, or at least 90% identical, and most preferably at least 95% identical to a native sequence. Such analogs must retain the biological activity of the native IFN sequence or fragment thereof, or possess antagonistic activity as described herein below.

The term "sequence identity" is used herein to refer to the following sequences in comparison. These sequences were aligned using the GAP (global alignment memt program) version 9 arrangement of the Genetic Computing Group, using the default (BLOSUM62) matrix (values-4 to +11), space penalty-12 (for the first gap of a space), and space extension penalty-4 (every successive gap added to the space). Percent identity is calculated after alignment and is expressed as the number of matched amino acids as a percentage of the number of amino acids in the claimed sequence.

The congeners of the invention can also be determined as follows. For the mifpar 2 portion of the complex, or the IFN portion thereof, the DNA and IFN sequences of IFNAR previously known in the art can be found in the literature cited in the background section of the specification, or readily located by one of ordinary skill in the art. Nucleic acids, such as those encoded by DNA or RNA that hybridize to the complement of native DNA or RNA under high or moderate stringency conditions, are also considered to be within the scope of the invention, as long as the polypeptide retains the biological activity of the native sequence, or in the case of IFN, the biological activity or antagonistic activity of MIFNAR2 or MIFNAR 2-EC. "stringent conditions" refers to conditions for hybridization and subsequent washing, which are routinely used by those of ordinary skill in the art and are referred to as "stringent". See Ausubel et al, Current protocols Molecular Biology, supra, Interscience, N.Y., § 6.3 and 6.4(1987, 1992) and Sambrook et al (Sambrook, J.C., Fritsch, E.F., and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Without limitation, examples of stringent conditions include washing conditions: hybridization temperatures Tm calculated at the time of study were 12-20 ℃ below washing with, for example, 2 XSSC and 0.5% SDD for 5 minutes, 2 XSSC and 0.1% SDD for 15 minutes, 0.1 XSSC and 0.5% SDD for 30-60 minutes at 37 ℃, and then 0.1 XSSC and 0.5% SDD for 30-60 minutes at 68 ℃. One of ordinary skill in the art will also recognize that stringency conditions will also depend on the length of the DNA sequence, oligonucleotide probe (e.g., 10-40 bases), or mixed oligonucleotide probe. If a mixed probe is used, trimethyl ammonium chloride (TMAC) is preferably used in place of SSC. See Ausubel, supra.

"functional derivatives" as used herein includes derivatives prepared from functional groups on the side chains of residues, or the N-or C-terminal groups, by methods known in the art, as long as they are still pharmaceutically acceptable, i.e., they do not destroy the biological activity of the corresponding part of the complex described herein, do not cause toxicity to the composition containing them or to the complex prepared above, and are included in the present invention. Derivatives may be chemical substances such as carbohydrate or phosphate residues, as long as such components have the same biological activity and remain pharmaceutically acceptable.

For example, derivatives may include fatty esters of the carboxyl terminal carboxyl group, amides of the carboxyl group by reaction with ammonium or with primary or secondary amines, N-acyl derivatives or free amino groups of amino acid residues with acyl molecules (e.g., alkyl or carbocyclic aryl), or O-acyl derivatives of free hydroxyl groups (e.g., seryl or threonyl residues) with acyl molecules. Such derivatives may also include, for example, polyethylene glycol side chains, which may mask antigenic sites, prolonging retention of the complex or radical active moiety in body fluids.

The term "fusion protein" refers to a polypeptide comprising MIFNAR2 or MIFNAR2-EC or analogs or fragments thereof fused to another protein, e.g., to increase residence time in body fluids. MIFNAR2 or MIFNAR2-EC may be fused to another protein, polypeptide, etc., such as an immunoglobulin or fragment thereof.

The fragment of the invention may be, for example, a fragment of MIFNAR2 or MIFNAR 2-EC. The term fragment refers to any subgroup of this molecule, i.e. shorter peptides that retain the desired biological activity. Such fragments can be readily prepared by removing some of the amino acids from one of the two ends of the MIFNAR2 molecule and testing the resulting fragments for the ability to bind IFN- β. The use of proteases to remove one amino acid at a time from one of the N-or C-termini of known polypeptides to determine whether these fragments retain the desired biological activity involves only routine experimentation.

As the active fragment of MIFNAR2, its analogs and fusion proteins, the present invention also includes the related molecules or residues thereof, such as fragments or precursors of sugar or phosphate residues, or aggregates of the protein molecules or sugar residues themselves, either alone or linked thereto, so long as the fragments have substantially similar activity.

The term "salt" as used herein refers to a carboxylic salt or acid addition salt of a complex of the invention, or an analogue thereof. Salts of the carboxyl groups may be formed by methods known in the art and include inorganic salts such as sodium, calcium, ammonium, iron or zinc salts and the like, salts with organic bases such as amines, triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as hydrochloric acid or sulfuric acid, and salts with organic acids such as acetic acid or oxalic acid. Of course, these salts must have substantially similar biological activity to the complexes of the invention or their analogs.

The term "biologically active" is explained herein below. To date, with regard to mifpar 2, an important biological activity is the ability to bind IFN- β with high affinity. It is therefore necessary to select those analogs, or variants, salts and functional derivatives that retain this interferon binding ability. This ability can be determined using conventional binding assays. Furthermore, fragments of mifpar 2 or analogs thereof may also be used, provided that they retain their interferon binding capacity. Such fragments can be readily prepared by removing the amino acid at one of the termini of the interferon-binding peptide and testing the resultant for interferon-binding properties.

In addition, polypeptides having interferon binding activity, i.e., MIFNAR2, MIFNAR2-EC, analogs, functional derivatives thereof may also contain additional amino acid residues flanking the interferon binding polypeptide. If the resulting molecule retains the enhanced interferon-binding ability of the core polypeptide, routine experimentation can be used to determine whether these flanking residues affect the basic and novel characteristics of the core peptide, i.e., its interferon-binding characteristics. The term "substantially identical" when used in reference to a particular sequence means that the additional flanking residues do not affect the basic and novel features of the particular sequence. This term does not include substitutions, deletions or additions in the particular sequence.

Although MIFNAR2 or MIFNAR2-EC has been used in the description and examples herein, it should be understood that this is only a preferred example and that the IFNAR1 subunit, and in particular the extracellular domain thereof, may be used with MIFNAR2 or MIFNAR 2-EC.

With respect to the interferon portion of the complexes of the invention, it is desirable to utilize the dependent interferon activity that any analog, functional derivative, fusion protein or fragment must retain biological activity. In most cases, this is the ability to bind to native cell surface receptors, whereby the receptors mediate signal generation. Thus, any such congeners, derivatives or fragments should retain such receptor agonistic activity useful in the present invention. On the other hand, molecules that antagonize this receptor may sometimes be used to prevent the biological activity of the natural interferon. Such antagonists may also be useful in prolonging the beneficial effects of the complexes of the invention. For example, may be used in situations where it is desired to eliminate the adverse effects of interferon, and analogs which, while still bound by the receptor and IFNAR portion of the complex, are unable to mediate signaling of the receptor by the native interferon and block signal production (i.e., interferon antagonism), are considered to be biologically active in accordance with the present invention and are encompassed by the term interferon as used in the complexes of the invention. Direct assays can determine whether such analogs retain the agonist activity of the receptor or have receptor antagonist activity and are therefore useful in one of the uses of the invention.

The invention also relates to a sequence encoding MIFNAR2EC, such as a sequence encoding SEQ ID NO: 2. 3 and 4, or analogs and fragments thereof, and DNA vectors carrying such DNA sequences, for expression in a suitable prokaryotic or eukaryotic host cell.

The ability to use recombinant protein expression profiles to produce large quantities of heterogeneous proteins has led to the development of various therapeutic agents, such as t-PA and EPO (Edington, 1995). Various hosts for the expression of recombinant proteins can be produced from prokaryotic origins (e.g.bacteria) (Olins, 1993), lower eukaryotes (e.g.yeast, Ratner, 1989) to higher eukaryotes (e.g.insect and mammalian cells, Reuveny, 1993; Reff, 1993). All these systems rely on the same principle-introducing the DNA sequence of the protein of interest into the selected cell type (in transient or stable form, as integrated or episomal elements) and using the host's transcription, translation and transport mechanisms to overexpress the introduced DNA sequence as a heterogeneous protein (Keown, 1990).

Various Protocols for the production of recombinant heterologous proteins have been reported (Ausubel et al, Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, NY, 1987-1995; Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).

In addition to expressing the native gene sequence, the ability to manipulate DNA at the nucleotide level has prompted the development of new engineered sequences that, although based on the native protein, have new activities due to changes in the primary structure of the protein (Grazia, 1997).

In addition, selected DNA sequences can be physically ligated to generate transcripts that can be developed into new fusion proteins that become a polypeptide once the independent protein is expressed (Ibanez, 1991). The activity of such fusion proteins may vary, e.g., be stronger than either of the respective proteins (Curtis, 1991).

For co-administration of MIFNAR2-EC and IFN, human IFN- β can be produced from a production process using mammalian Chinese hamster ovary Cells (CHO), as disclosed in EP 220574. Type I interferons can be expressed in a variety of host cells, including bacterial (Utsumi, 1987), insect (Smith, 1983) and human (Christofinis, 1981) cells. Human mifmar 2 or fragments thereof may also be expressed in CHO host cells. For secretion of MIFNAR2-EC from CHO cells, the MIFNAR2-EC DNA sequence can be ligated with the sequence of the human growth hormone signal peptide, as described in patent application WO 0022146. Alternatively, soluble receptors can be successfully expressed in bacterial expression systems, such as MIFNAR2-EC (Terlizze, 1996).

The invention also relates to a pharmaceutical composition comprising as active ingredient mifpar 2, mifpar 2-EC, mifpar 2-EC/IFN complex or their congeners, fusion proteins, functional derivatives, fragments thereof; or mixtures thereof; or a salt thereof; and a pharmaceutically acceptable carrier, diluent or excipient. Embodiments of the pharmaceutical compositions of the present invention include those that enhance the effects of IFN in the treatment of viral diseases, anticancer therapy, immunomodulatory therapy, such as autoimmune diseases, and other applications associated with interferons and cytokines.

The pharmaceutical compositions of the invention for administration may be prepared by mixing MIFNAR2, MIFNAR2-EC, MIFNAR2-EC/IFN complexes or their congeners, fusion proteins, functional derivatives, fragments thereof; or mixtures thereof; or a salt thereof; mixing with physiologically acceptable stabilizer and/or excipient. The dosage form is for example a vial lyophilized dosage form. The method of administration may be any of the accepted modes of administration of similar drugs and depends on the disease to be treated, for example, intravenous, intramuscular, subcutaneous, local injection or topical application, or continuous infusion, etc. The amount of active compound administered depends on the route of administration, the disease to be treated and the condition of the patient.

The present invention relates to a method of treating autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, lupus and ulcerative colitis, which comprises administering a therapeutically effective amount of mifar 2, mifar 2-EC, mifar 2-EC/IFN complex or their congeners, fusion proteins, functional derivatives, fragments thereof; or mixtures thereof; or a salt thereof.

The present invention also relates to a method of treating viral diseases such as granulomatous diseases, condyloma acuminatum, juvenile laryngeal papillomatosis, chronic infections caused by hepatitis a or b and c viruses, which comprises administering a therapeutically effective amount of mifmar 2, mifmar 2-EC, mifmar 2-EC/IFN complex or their congeners, fusion proteins, functional derivatives, fragments thereof; or mixtures thereof; or a salt thereof.

The present invention also relates to a method of treating various types of cancer, such as hairy cell leukemia, Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkin's lymphoma or melanoma, comprising administering a therapeutically effective amount of MIFNAR2, MIFNAR2-EC, MIFNAR2-EC/IFN complex or their congeners, fusion proteins, functional derivatives, fragments thereof; or mixtures thereof; or a salt thereof.

In the above method, MIFNAR2, MIFNAR2-EC, MIFNAR2-EC/IFN complex or their congeners, fusion proteins, functional derivatives, or fragments thereof; or mixtures thereof; or a salt thereof, may be administered with an IFN, preferably IFN- β.

"therapeutically effective amount" means the amount of mifpar 2, mifpar 2-EC, mifpar 2-EC/IFN complex or their congeners, fusion proteins, functional derivatives, fragments thereof, after administration; or mixtures thereof; or a salt thereof, will result in modulation of the biological activity of IFN- β. The dosage administered to an individual as a single dose or multiple doses will vary depending on a variety of factors, including the route of administration, the condition and characteristics of the patient (sex, age, weight, health, size), the extent of the condition, concurrent therapy, the frequency of therapy, and the desired effect. The dosage ranges set can be adjusted and manipulated within the ability of the person skilled in the art, and the mifpar 2, mifpar 2-EC, mifpar 2-EC/IFN complex or their congeners, fusion proteins, functional derivatives, fragments thereof are determined; or mixtures thereof; or salts thereof, in vitro and in vivo.

Local injections, for example, will be less than intravenous infusions depending on body weight requirements.

Free IFN- β has a tendency to oligomerize. To suppress this tendency, current IFN- β formulations have an acidic pH and thus may cause some local irritation when administered. Due to MIFNAR2, MIFNAR2-EC or their congeners, fusion proteins, functional derivatives, fragments thereof; or mixtures thereof; or salts thereof, act as a super-stabilizer for the wild-type IFN- β factor and thus prevent its oligomerization, and their use in IFN- β preparations stabilizes IFN- β and thus eliminates the need for acidic preparations. Therefore, the protein contains MIFNAR2, MIFNAR2-EC or congeners, fusion proteins, functional derivatives and fragments thereof; or mixtures thereof; or salts thereof, with other conventional pharmaceutically acceptable excipients are also part of the invention.

The invention also relates to the use of MIFNAR2, MIFNAR2-EC, MIFNAR2-EC/IFN complexes or their congeners, fusion proteins, functional derivatives, fragments thereof; or mixtures thereof; or a salt thereof for antiviral, anticancer and immunomodulatory treatment. In particular, the mutant interferon receptor and interferon compound of the present invention can be used for antiviral treatment of diseases such as granulomatous diseases, condyloma acuminatum, juvenile laryngeal papillomatosis, chronic infection caused by hepatitis A or hepatitis B and hepatitis C virus

In particular, the mutant interferon receptor-interferon complex of the present invention can be used for anticancer therapy of diseases such as hairy cell leukemia, Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-Hodgkin's lymphoma or melanoma

The mutant interferon receptor and the compound of the mutant interferon receptor and the interferon can also be used for immunoregulation treatment of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, lupus and ulcerative colitis.

An "autoimmune disease" is a disease in which an individual's immune system begins to successfully fight its own body. The immune system produces antibodies against its own tissues. Virtually every part of the body is predisposed to have a difficult co-autoimmune disease.

Mutant interferon receptors, complexes of mutant interferon receptors with interferon, are also useful in neurodegenerative diseases, particularly multiple sclerosis.

The invention also relates to a pharmaceutical composition containing MIFNAR2, MIFNAR2-EC, MIFNAR2-EC/IFN complex or their congeners, fusion proteins, functional derivatives, and fragments thereof; or mixtures thereof; or its salt pharmaceutical composition, relate to and contain the expression vector, express MIFNAR2, MIFNAR2-EC, MIFNAR2-EC/IFN complex or their congeners, fusion protein, functional derivative, its slow virus gene therapy vector of fragment specifically.

The term "treating" as used herein is to be understood as preventing, inhibiting, attenuating, alleviating, or reversing any or all of the symptoms or causes of a disease.

Having now described the invention, the same will be more readily understood through the following examples which are given by way of illustration and are not intended to be limiting of the present invention.

Examples

Example 1: expression and purification of proteins

Escherichia coli-expressed IFNAR2-EC (extracellular domain) and IFN α were purified by ion exchange and size exclusion chromatography as described (Piehler & Schreiber, 1999A). IFNAR2-EC mutant expression level and wild type as high. Wild-type glycosylated IFN- β was produced in CHO (as described in EP 220574). Protein concentration was determined from the absorbance at 280nm (Piehler & Schreiber, 1999A) for IFN α 2 at 1:280 ═ 18,070M-1; 1:280 ═ 30,050M-1 for IFN- β; for IFNAR2-EC 1:280 ═ 26,500M-1 (for IFNAR2-EC tryptophan mutants of W102A and W74F, the correction is 1:280 ═ 21, 100M-1). Protein purity was analyzed by SDS-PAGE under non-reducing conditions.

Example 2: generation of IFNAR-EC mutants

Site-directed mutagenesis was performed using the template pT72CR2(Piehler & Schreiber, 1999A) and nucleotide primers containing mutated codons as detailed in (Albeck & Schreiber, 1999) using high fidelity polymerase pwo (Boehringer Mannheim) and pfu (Stratagene) as PCR. After phosphorylation and ligation, E.coli TG1 cells were transformed with the mutated plasmid. The entire sequence of the expressed gene containing the mutation was verified by DNA sequencing (Ausubel et al, Current protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, NY, 1987-1995; Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).

The resulting mutants have two amino acid residues: histidine 78 (H78) and asparagine 100 (N100) were mutated: a-is changed to alanine both (H78A/N100A), B-is changed to alanine and aspartic acid respectively (H78A/N100D), and C-is changed to alanine and histidine respectively (H78A/N100H).

Example 3: thermodynamic and kinetic analysis

All thermodynamic and kinetic data were obtained from the free-labeled hybrid phase detection. The interaction between IFN-. beta.2 and IFNAR2-EC was monitored by reflectance interference spectrophotometry [ rifS ] under flow-through conditions as described by Piehler & Schreiber, 1999A. This method is similar to Biacore and is used to accurately determine the affinity of binding between two proteins. IFNAR2-EC (wild type or mutant) was immobilized by immobilized specific antibody (as described by Piehler & Schreiber, 2001). All assays for IFN-. beta.IFN-. alpha.2 and IFNAR2-EC were performed in 50nM Hepes and 500mM NaCI, 0.01% TritonX100, pH 7.4. The interaction at 500mM NaCI was determined to eliminate the non-specific reaction of IFN- β with the surface seen at 150mM NaCI.

Binding and separation kinetics were determined with standard injection protocols and corrected with blank runs. The dissociation rate constant at 1-1000nM IFN concentration was determined to saturate the surface. The 1:1 kinetic model was fitted with the entire separation range (Piehler & Schreiber, 2001).

Example 4: test for antiviral Activity

IFN- β antiviral activity was measured to inhibit the cytopathic effects of Vesicular Stomatitis Virus (VSV) on human WISH cells (Rubinstein et al, 1981).

Example 5: determination of IFN binding to mutant IFNAR2

IFN- β and IFN α 2 binding to the H78A/N100A mutant was determined and compared to the wild-type EC receptor using rifS (example 3). Although IFN- β was found to bind to the H78A/N100A mutant at a similar rate to the wild type (FIG. 3), the off-rate was found to be significantly lower. The calculated affinity of IFN- β to the H78A/N100A mutant was about 30pM and to the wild-type protein was about 3 nM. In contrast to IFN- β, IFN α 2 binding to H78A/N100A mutant and dissociation rates were found to be similar to those obtained with the wild-type protein (FIG. 3). These results show that IFNAR2 mutant affinity than the wild type for IFN beta about 100 times higher, for IFN alpha 2 is unchanged.

Example 6: relative affinity of interferon to mutant IFNAR2

IFNAR2 wild type or mutant immobilized on the surface through specific antibodies (example 3), IFNAR-EC receptor and mutant receptor EC (example 2) on IFN beta and IFN alpha 2 binding and affinity. After determining the affinity, relative affinities were obtained by comparing the kd of the mutant receptor with the kd of the wild-type receptor (table 4).

The Kd for binding of interferon to the extracellular domain (EC) of IFNAR2 was measured by RIfS and found to be about 3nM (example 5). IFN beta and H78A/N100A (EC) mutant combined with the Kd of about 30 pM. Accurate measurement of Kd for this mutant is not possible because the binding is so tight that good data cannot be obtained from RIfS. IFN alpha 2 and H78A/Nl00A EC mutant Kd and wild type receptor similarity. The results in table 4 show the relative affinity of IFNAR EC mutants with respect to the wild-type IFNAR2 receptor EC. The mutants are as follows: mutation at one amino acid residue H78A or N100A and into two amino acids H78A/N100A, H78A/N100D and H78A/N100H, wherein the amino acid N100 is mutated to alanine, aspartic acid or histidine, respectively (example 2). The results confirmed that a single mutation in IFNAR2 increased the affinity of the complex by 4.6-7.3 fold, while the double mutation caused a synergistic effect, increasing the affinity of the complex by 26-50 fold. From the affinity point of view, the best mutant was found to be a double mutation modifying N100 to alanine, which increased the affinity by 50-fold compared to the wild type.

IFNAR2	IFNα2	IFN-β
			Wild type	1.0	1.0
H78A	0.4	4.6
			N100A	2.0	7.3
H78A/N100A	0.7	>50
			H78A/N100D	1.0	40.0
H78A/N100H	0.9	26.0

TABLE 4

Example 7: IFNAR2 mutant blocking interferon IFN-beta

IFNAR2-EC wild type and mutant EC as IFN beta dynamic vector ability comparison. For this purpose, samples containing a constant concentration of IFN- β (10pM) were monitored for residual (free) IFN- β antiviral activity after mixing with different concentrations of recombinant soluble IFNAR2-EC or IFNAR2 mutant EC (example 6). In this antiviral assay, the mixture (IFNAR2/IFN) was added to WISH cells (human amniotic cells). These WISH cells were then challenged with Vesicular Stomatitis Virus (VSV) and the antiviral activity of residual (free) IFN- β was monitored as cell viability 24 hours after inoculation (example 4). From the antiviral activity survival dose curve determination of the sample in the presence of free IFN- β, with different amount of wild type or mutant IFNAR2-EC (R2) concentration, as in the absence of IFNAR2 determination of IFN- β concentration content (figure 2 upper panel).

The mutants tested were as follows: IFNAR2-EC mutated at one amino acid residue H78A or N100A and at two amino acids H78A/N100A, H78A/Nl00D and H78A/N100H, in which the amino acid N100 is mutated to alanine, aspartic acid and histidine, respectively (example 2). IFNAR 2H78A/N100A double mutant (example 2) showed the highest affinity among all the mutants produced (kd about 30pM or less, see examples 5 and 6).

FIG. 4 shows that about 20% of IFN- β binds to soluble receptors (blocks) in the presence of 2.5nM wild type IFNAR2-EC, whereas 50% of IFN- β binds in the presence of the double mutant EC H78A/N100A at only 0.2nM, and 80% of IFN- β binds with only 0.4nM of the double mutant EC H78A/N100A. Biological experiments also demonstrated that the same degree of IFN- β blockade (bound IFN- β under equilibrium conditions) and residual antiviral activity (free IFN- β) obtained with wild-type IFNAR2 can be achieved with the H78A/N100A IFNAR2 mutant EC at about 30-fold lower concentrations. This result also shows that double modified mutants produced the best results, especially H78A/N100A with two amino acid mutations to alanine confirmed IFNAR 2.

This result shows that the double mutant IFNAR2 did block IFN- β more effectively, thus requiring lower amounts to achieve its carrier activity.

Reference to the literature

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Sequence listing

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Claims (37)

1. Use of a type I interferon receptor 2 mutant polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 at amino acid residue 78 and asparagine at position 100, wherein the IFNAR2 mutant polypeptide binds interferon beta with 15-100 times greater affinity than the wild-type protein.
2. 2. The use of claim 1, wherein the mutation is a substitution.
3. 3. The use of claim 2, wherein the substitution is non-conservative.
4. 4. The use of claim 1, wherein histidine 78 is replaced by alanine.
5. 5. The use of claim 1, wherein the asparagine residue at position 100 is replaced with alanine, aspartic acid or histidine.
6. 6. The use of claim 4, wherein residues 78 and 100 are replaced by alanine.
7. 7. The use of claim 5, wherein residues 78 and 100 are replaced by alanine.
8. 8. The use of claim 1, wherein the IFNAR2 mutant polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 2, SEQ ID NO: 3 or SEQ ID NO: 4.
9. 9. the use of claim 1, wherein said IFNAR2 mutant polypeptide has an affinity for IFN- β of about 30 pM.
10. 10. The use of claim 1, wherein the IFNAR2 mutant polypeptide has an affinity for IFN- β that is about 25-fold greater than the affinity of the wild-type IFNAR 2.
11. 11. The use of claim 1, wherein the IFNAR2 mutant polypeptide has an affinity for IFN- β that is about 50-fold greater than the affinity of the wild-type IFNAR 2.
12. 12. The use of claim 1, wherein the IFNAR2 mutant polypeptide has an affinity for IFN- β that is about 100-fold greater than the affinity of the wild-type IFNAR 2.
13. 13. The use according to claim 1, wherein the IFNAR2 mutant polypeptide is covalently bound to IFN.
14. 14. The use according to claim 13, wherein the IFN is IFN- β.
15. 15. The use of claim 1, wherein said IFNAR2 mutant polypeptide is pegylated.
16. 16. The use of claim 1, wherein the medicament further comprises IFN.
17. 17. The use of claim 16, wherein the IFN is IFN- β.
18. 18. The use of claim 1, wherein the medicament further comprises an IFN antagonist.
19. 19. The use according to claim 1, wherein the medicament is a medicament for increasing IFN activity.
20. 20. The use according to claim 19, wherein the medicament is a medicament for increasing the anti-cancer activity of IFN.
21. 21. The use according to claim 19, wherein the medicament is a medicament for enhancing the immunomodulatory therapeutic properties of IFN.
22. 22. The use according to claim 21, wherein the medicament is a medicament for increasing the immunomodulatory activity of IFN in autoimmune diseases selected from the group consisting of multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, lupus and ulcerative colitis.
23. 23. The use according to claim 1, wherein the medicament is a medicament for inhibiting IFN activity.
24. 24. A pharmaceutical composition comprising a therapeutically effective amount of a type I interferon receptor 2 mutant polypeptide, said IFNAR2 mutant polypeptide consisting of SEQ ID NO: 1 at amino acid residue 78 and asparagine at position 100, wherein the IFNAR2 mutant polypeptide binds interferon beta with 15-100 times greater affinity than the wild-type protein.
25. 25. A pharmaceutical composition comprising a therapeutically effective amount of a fusion protein comprising a type I interferon receptor 2 mutant, said IFNAR2 mutant consisting of SEQ ID NO: 1 at amino acid residue 78 and asparagine at position 100, wherein the IFNAR2 mutant binds interferon beta with 15-100 times greater affinity than the wild-type protein.
26. 26. The pharmaceutical composition of claim 24 or 25, further comprising IFN.
27. 27. The pharmaceutical composition of claim 26, wherein the IFN is IFN- β.
28. 28. The pharmaceutical composition of claim 24 or 25, further comprising an IFN antagonist.
29. 29. The pharmaceutical composition of claim 27, wherein said IFNAR2 mutant polypeptide is covalently bound to IFN- β.
30. 30. The use according to claim 1, wherein the medicament is a medicament for the treatment of an autoimmune disease, a viral disease or cancer.
31. 31. The use of claim 30, wherein the disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, myasthenia gravis, diabetes, lupus, ulcerative colitis, chronic granulomatous disease, condyloma acuminatum, juvenile laryngeal papillomatosis, hepatitis a, hepatitis b, chronic infection by hepatitis c virus, hairy cell leukemia, kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia, non-hodgkin's lymphoma and melanoma.
32. 32. The use of claim 30, wherein the medicament further comprises a therapeutically effective amount of IFN- β.
33. 33. The use of claim 31, wherein the medicament further comprises a therapeutically effective amount of IFN- β.
34. 34. The use according to claim 1, wherein the medicament is a medicament for the treatment of a disease caused or exacerbated by IFN- β.
35. 35. The use of claim 34, wherein the medicament further comprises an IFN- β antagonist.
36. 36. The use according to claim 1, wherein the medicament is an agent for preventing IFN oligomerization.
37. 37. The use according to claim 36, wherein the medicament is an agent for preventing IFN- β oligomerization.